The Oxygen Release Instrument: Space Mission

life

Article The Oxygen Release Instrument: Space Mission Reactive Oxygen Species Measurements for Habitability Characterization, Biosignature Preservation Potential Assessment, and Evaluation of Human Health Hazards

1, 2 3 1, Christos D. Georgiou *, Christopher P. McKay , Richard C. Quinn , Electra Kalaitzopoulou †, 1, 1, Polyxeni Papadea † and Marianna Skipitari † 1 Department of Biology, University of Patras 26504, Greece 2 NASA Ames Research Center, Moﬀett Field, CA 94035, USA 3 SETI Institute, Carl Sagan Center, Mountain View, CA 94043, USA * Correspondence: [email protected]; Tel.: +30-2610-997-227; Fax: +30-2610-969-278 These authors contributed equally to this work. † Received: 29 May 2019; Accepted: 25 August 2019; Published: 27 August 2019

Abstract: We describe the design of an instrument, the OxR (for Oxygen Release), for the enzymatically specific and non-enzymatic detection and quantification of the reactive oxidant species (ROS), 2 superoxide radicals (O2•−), and peroxides (O2 −, e.g., H2O2) on the surface of Mars and Moon. The OxR instrument is designed to characterize planetary habitability, evaluate human health hazards, and identify sites with high biosignature preservation potential. The instrument can also be used for missions to the icy satellites of Saturn’s Titan and Enceladus, and Jupiter’s Europa. The principle of the OxR instrument is based on the conversion of (i) O2•− to O2 via its enzymatic dismutation (which also releases H2O2), and of (ii) H2O2 (free or released by the hydrolysis of peroxides and by the dismutation of O2•−) to O2 via enzymatic decomposition. At stages i and ii, released O2 is quantitatively detected by an O2 sensor and stoichiometrically converted to moles of O2•− and H2O2. A non-enzymatic alternative approach is also designed. These methods serve as the design basis for the construction of a new small-footprint instrument for specific oxidant detection. The minimum 2 detection limit of the OxR instrument for O2•− and O2 − in Mars, Lunar, and Titan regolith, and in Europa and Enceladus ice is projected to be 10 ppb. The methodology of the OxR instrument can be rapidly advanced to flight readiness by leveraging the Phoenix Wet Chemical Laboratory, or microfluidic sample processing technologies.

Keywords: planetary oxygen-based reactive oxidants; instrument; habitability; biosignatures

1. Introduction On Earth, the production of reactive oxygen species (ROS) in soils is typically associated with the relatively high abundance of O2(g) in the atmosphere [1]. In other solar system environments, or space environments beyond our solar system, where O2(g) exists only in trace amounts (e.g., Mars [2], the Earth’s Moon [3,4], Europa [5], Saturn’s rings [6], interstellar clouds [7]) the production and accumulation of ROS is not precluded. Even in planetary environments lacking O2(g), ROS can be produced by many well-known natural processes, for example, environments containing H2O, CO, and/or CO2 [8]. On the Moon (and presumably on Mars), ROS can be generated by the interaction of H2O ice with cosmic rays [9]. Experiments indicate that Lunar (and presumably Martian) dust can generate hydroxyl free radicals (•OH) via the Fenton reaction as demonstrated with Lunar simulants [10] and Fe-rich silicate

Life 2019, 9, 70; doi:10.3390/life9030070 www.mdpi.com/journal/life Life 2019, 9, 70 2 of 16

minerals [11]. Freshly fractured Lunar regolith can produce large amounts of H2O2 and other ROS [12], which are considered to play a role in Lunar dust toxicity [13]. Although, none of the curated Apollo mission Lunar samples exist in a state that fully preserves the reactive chemical surfaces aspects (i.e., ROS) that would be expected to be present on the lunar surface, freshly ground Lunar + soil has been shown to produce •OH upon contact with H2O[14]. On Mars, reactive O2 and O2− can form through the release of reactive oxygen via scattering of CO2 ions from solid surfaces; where oxygen produced is preferentially ionized by charge transfer from the surface over the predominant atomic oxygen product [8]. ROS may also be produced by Martian regolith via silicate abrasion during dust storms [15] (e.g., by mechano-radical production [16]). Silica fracturing is known to generate + surface free radicals (homolytic and heterolytic fracturing form Si•/SiO•, and Si /SiO−, respectively), which upon reaction with H2O or H2O2 generate •OH [17]. Such dust/silica-induced radicals may pose a serious human health hazard (verified by toxicity studies on mammalian cells [18]), during future manned missions to Mars and Moon. Beyond Mars and the Moon, complex interactions between Saturn and its satellites Titan and Enceladus can cause the generation and movement of oxygen from the latter to the former [19,20]. Ice water from Enceladus south polar plumes can be radiolytically oxidized to H2O2 and O2, by energetic particles from Saturn’s radiation belts (mostly electrons). Such ROS emanating from this radiolytic gas-driven cryovolcanism can be continuously accumulated deep in icy regolith [19]. Concurrently, H2O molecules escaping from Enceladus’ plumes should be split by magnetospheric plasma (protons, + + H 2, water group ions) into neutral and charged particles (O ), which can enter Titan’s atmosphere + and be captured by fullerenes (a hollow carbon atom shell, e.g., of C60). Exogenic keV O could become free oxygen within those fullerene aerosols, and eventually, fall free onto Titan’s surface. Such a process could be driven by cosmic ray interactions with aerosols at all heights, and can eventually, drive pre-biotic chemistry [20]. It has been suggested that ice-covered worlds require an external source of oxidants to maintain biological viability [21]. Hand et al. 2007, have proposed that oxidants produced by UV and ionizing radiation on the surface of icy worlds, such as Europa, can be carried down to the water column to react with reduced species to provide a source of redox energy [22]. In light of all these considerations, measurement of planetary ROS is of great interest for astrobiology, including the exploration of Titan, Enceladus, and Europa, and important for human missions to the Moon and Mars. However, instruments for the in situ specific detection of the key ROS 2 O2•− and O2 − (e.g., H2O2) in these extreme environments have not yet been developed. The justification for this type of instrument is supported by the results of the Viking Mars mission. In 1976, the Viking Lander performed biological experiments designed to detect extant life in Martian regolith. The reactivity of the Martian regolith was first indicated by the release of O2 in the Gas Exchange Experiment (GEX), and the decomposition of organics, contained a culture media, in the Labeled Release (LR) experiment [23–25]. In the GEX, up to ~770 nmoles O2(g) was produced from -3 regolith samples (1 cm ) upon humidification or wetting. The persistence of O2(g) release from samples that were heated to 145 ◦C for 3 h and then cooled prior to wetting or humidification, ruled out a biological explanation of the GEX results [23,26]. In the Viking LR, up to ~30 nmoles 14C labeled -3 gas, presumed to be CO2, was released after regolith samples (0.5 cm ) were wetted with an aqueous solution containing 14C-labeled organics [27,28]. The release of 14C-labeled gas in the LR was eliminated by heating the sample to 160 ◦C for 3 h and then cooling prior to the addition of the labeled aqueous organics. These results lead to the conclusion that the Martian surface material contains more than one type of reactive oxidants [23]. Metal salts of O2•− were among the earliest proposed explanations for the thermally stable agent responsible for O2(g) release in the GEX. In the case of the LR, peroxide was 14 among the earliest explanations proposed for the thermally liable agent responsible for CO2 release. In addition to the possible presence of metal salts of O2•−, it has been proposed that O2•− is generated on Martian dust and regolith surfaces by a UV-induced mechanism [29]. Such a mechanism for O2•− photo-generation has also been shown with Mars analog Mojave and Atacama regolith [1]. Life 2019, 9, 70 3 of 16

More recently, high levels of regolith perchlorate (ClO4−) were directly detected at the Phoenix landing site [30]. Following up on this result, the presence of ClO4− at the Viking landing sites was inferred [31], and its presence at the Martian equator verified by the Sample Analysis at Mars (SAM) instrument on Mars Science Laboratory (MSL)-based on thermal analyses [32]. While the stability of ClO4− under the conditions of the GEX and LR preclude it as a direct explanation for these experiments, it has been suggested that ClO4− radiolysis products reproduce the major aspects of both experiments [25]. The form of the trapped O2, in particular, derived from ClO4− radiolysis, was not identified, and it has been suggested that some fraction may exist as superoxide radical or peroxide [25,33]. This suggestion was confirmed by the finding that both of these oxidants are generated—together with •OH—by γ-ray exposure of ClO4− (mixed in Mars salt analogs) upon water wetting [34]. Preceding this finding, other possible oxidants present on the surface of Mars have been reviewed in detail [35]. In the context of instrument development for in situ analysis, it is useful to note that the expected concentration of oxidants, as inferred from the Viking Biology Experiments, is at the parts per million level (Table1 in [36]). Given the poorly understood nature and distributions of oxidants in Martian and Lunar regolith, there is a need for the development of flight instruments for their specific identification and quantification. The only flight instrument previously built for the quantitative, although non-specific, in situ determination of oxidants was the Mars Oxidant Experiment (MOX) instrument [36] as the United States contribution to the failed Soviet Union’s Mars ’96 mission. That instrument would have exposed materials-sensors to the Martian regolith and monitored their reaction with oxidants over time. Materials included various metallic (e.g., Al, Ag, Pb, Au) and organic layers (e.g., L- and D-cysteine). 2 New developments have made possible the detection of reactive oxidants, such as peroxides (O2 −), O2•−, and •OH [1,34]. In planetary and terrestrial regolith, O2•− may exist as adsorbed (O2•−ads)[1] or + n+ present in metal salts (Me O2•−), such as KO2 and NaO2 [37], and in ionic complexes with metals (Me 2 − O2•−) of certain minerals and oxides [38,39]. Metal peroxides can exist as salts of metals with O2 ¯ bonding 2+ 2 + 2 either as Me O2 − (e.g., CaO2, MgO2) or as Me 2O2 − (e.g., Na2O2,K2O2). Metal peroxides can also exist 4+ 4+ 4+ 2+ 2+ + + as hydroperoxides (MeO2H; e.g., of Ti , Zr , and Ce )[40]. The presence of Mg , Ca ,K , and Na ions on Martian regolith (measured with the Phoenix Mars Lander Wet Chemistry Lab [30,41,42]) and on Lunar regolith [43], may provide the needed counter ions for stabilization of metal salts of O2•−, peroxides, 2 and hydroperoxides in the regolith. Metal salts of O2•− and hydro/peroxides (i.e., O2 −) can undergo aqueous decomposition at neutral pH, releasing O2 and H2O2 (Table1).

2 Table 1. Aqueous decomposition of metal salts of O2•− and O2 −

Metal (Me) Salts of O2 Release O2 ( ) and H2O2 by the Following Reactions [37,44] •− ↑ Adsorbed O 2 O + 2 H O 2 OH + H O + O (1) 2•− 2•−ads 2 → − 2 2 2↑ 2 Me+ O + 2 H O 2 Me+OH + H O Metal salts of O 2•− 2 → − 2 2 (2) 2•− + O 2↑ 2 Men+ O + 2 H O 2 Men+ + 2 OH Metal-O complexes − 2•− 2 → − (3) 2•− + H O + O 2 2 2↑ Metal Peroxides/Hydroperoxides Release H2O2 by the Following Reactions [37] Me+ O 2 + 2 H O 2 Me+OH + H O (4) Me-salts of O 2 2 2 − 2 → − 2 2 2 − Me2+O 2 + 2 H O Me2+(OH ) + H O (5) 2 − 2 → − 2 2 2 Me-hydroperoxides (MeO H) MeOOH + H O MeOH + H O (6) 2 2 → 2 2

2. Principle of Operation of the OxR (for Oxygen Release) Instrument

2 The OxR instrument for the detection and quantiﬁcation of O2•− and O2 − addresses priorities for human exploration of Mars and the Moon as highlighted in the NASA plan to “Explore Moon to Mars” which will use the Moon as “a testbed for Mars [ ... ] and beyond.” [45]. Our approach is to quantitatively convert peroxides and superoxide radicals into O2(g), which can then be detected easily,precisely,and with very high sensitivity. The principle of the OxR instrument is based on the enzymatic conversion of the dismutation and hydrolysis products of superoxide radicals (O2•− adsorbed on Life 2019, 9, 70 4 of 16

2 mineral surfaces, O2•−ads, or released by the dissociation of metal salts of O2•−) and peroxide (O2 − as H2O2 or released by the hydrolysis of metal peroxides) to O2, followed by quantitative detection using an O2 electrode. The OxR instrument design includes a sealable, temperature- and pressure-controlled sample chamber. The chamber is equipped with an O2-sensor, and inlets for the sequential dispensing of three reagents, after each of which the concentration of released O2 is measured. The two enzymatic reagents (Cu/Zn-superoxide dismutase, SOD, and catalase, CAT) used are stored in a solid form separated from their aqueous solvents (to withstand cosmic radiation exposure). The third reagent, acetonitrile (ACN), is separately stored at any temperature above its melting point ( 46 C). The enzymic reagents are mixed − ◦ with their solvents right before use either by storing them in separate reagent crucibles (analogous to those used in the Wet Chemistry Laboratory of the 2007 Phoenix Mars Scout Lander mission), or in (commercially available) dual-chamber pre-ﬁllable syringes (one chamber for storing the enzyme reagent in solid form, and the other for its solvent, to be mixed upon piston movement), and their sequential dispensing in the chamber. The OxR instrument can detect released O2 by electrochemical or solid state optical O2-sensing electrodes. Both electrode types are commercially available. Optical O2-sensing electrodes are based on the luminescence quenching by O2, and are sensitive enough to 3 measure O2 at ~1 nmole O2 per cubic cm (cm− ) of regolith or water, i.e., much lower than that detected by the GEX (775 nmoles cm-3 regolith). This translates to a minimum instrument detection limit for 2 2 metal salts of O2•− and O2 − of 0.01 ppm (= 10 ppb) for Martian and Lunar regolith or O2 − in Europa 2 1 and Enceladus water. This sensitivity corresponds to ~10 µg O2•−/O2 − kg− Mars or Lunar surface 3 regolith (based on a density of 1.4–1.6 g cm− for Mars [46,47], and for the Moon [48]).

3. Enzyme-Based ROS Speciﬁcity of the OxR Instrument

We have developed an enzymatic methodology (OxR assay) for the detection of total O2•− (the sum of O , Me+ O , and Men+ O ) and total O 2 (the sum of Me2+O 2 , Me+ O 2 , and MeO H), 2•−ads 2•− − 2•− 2 − 2 − 2 2 − 2 for terrestrial field and planetary applications [49]. The use of enzymes for the OxR assay provides 2 specificity and quantification of regolith O2•− (reactions 1–3) and O2 − (reactions 4–6) through the measurement of the O2 that is enzymatically released during dismutation/hydrolysis. Specifically, this is achieved using the following enzymatic reactions [44]: (i) the SOD-catalyzed dismutation of 1 1 1 mole O2•− to ⁄2 mole O2 and ⁄2 mole H2O2, and (ii) the CAT-decomposition of 1 mole H2O2 (from 1 dismutated O2•− and hydrolyzed metal peroxides/hydroperoxides) to ⁄2 mole O2. The enzymatic reaction steps of the OxR assay have been established by the following experimental testing [49]: (i) The effective scavenging of O2•− via dismutation to O2 and H2O2 by SOD; (ii) the decomposition of H2O2 (from the hydrolysis of peroxides and the dismutation of O2•−) to O2 2 (by CAT) in the presence of ClO4− and carbonate (CO −; both Martian regolith constituents), and in potassium phosphate plus diethylene–triamine–pentaacetic acid (DTPA) buffer (pH 7.2). Phosphate is an H2O2-stabilizer [50], and DTPA acts as chelator of any soluble transition metal ions, which can destroy H2O2 via the Fenton reaction [51] and O2•− via oxidation to O2); (iii) the functional stability of the OxR assay enzymes SOD and CAT to cosmic rays upon exposure to γ-radiation; (iv) the simulation of the OxR assay by indirect testing on commercial analogues of metal salts of O2•− and 2 O2 −, and directly on O2•− and H2O2. The enzymatic (and accompanying non-enzymatic) reactions involved in the OxR assay are presented in Table2.

Table 2. Reactions of the OxR (Oxygen Release) assay.

+ + Step 1. Metal O2•− (e.g., Me O2•−) dissociation reaction: Me O2•− (in H2O) + → O2•− + Me Note: Stock solution of stable O is obtained by dissociation of Me+ O I. Metal O (O , Me+ O , Men+ 2•− 2•− 2•− 2•−ads 2•− − (e.g., KO ) in anhydrous acetonitrile (ACN). O ); additional details for their 2 2•− Step 2. Release of O (and H O ) via SOD-catalyzed dismutation of O (from I, hydrolysis/dismutation are presented 2 2 2 2•− step 1): 2 O2•− + 2 H2O 2 OH− + H2O2 + O2 (same as reaction 1) in reactions 1–3, and in Figure1 step a. → ↑ 5 Note: The spontaneous dismutation of O2•− by H2O has a rate constant ~2x10 M-1 s-1, while that with SOD is 32,000-fold faster; 6.4x109 M-1 s-1 [52]. Step 3. Base (MeOH) formation: Me+ + OH MeOH − → Life 2019, 9, 70 5 of 16

Table 2. Cont.

2 + 2 + 2 Step 1. Dissociation reaction of metal O2 − (e.g., Me 2O2 ¯): Me 2O2 − (in H2O) II. Metal O 2 (Me+ O 2 , Me2+O 2 , O 2 + 2 Me+ 2 − 2 2 − 2 − → 2 − MeOOH); additional details for their Step 2. Hydrolysis reaction of O 2 (from II, step 1): O 2 + 2 H O 2 OH + 2 − 2 − 2 → − hydrolysis are presented in reactions 4–6. H2O2 (same as reaction 4) Step 3. Base (MeOH) formation: 2 Me2+ + 2 OH 2 MeOH − → Release of O2 via CAT-catalyzed decomposition of H2O2 [44], resulting from I, III. H2O2 released by the hydrolysis of 2 step 2, and/or II, step 2: metalLife O20192•−, and9, x FOR O2 − PEER; additional REVIEW details are 6 of 16 2 H O 2 H O + O (7) shown in Figure1 step b. 2 2 → 2 2↑

FigureFigure 1. Simulation 1. Simulation of theof the OxR OxR (Oxygen (Oxygen Release) Release) assay on on O O2•2−•− andand H2 HO2:O It2 :is Itperformed is performed in the in the presencepresence/absence/absence of Mars-like of Mars-like regolith regolith from from Mojave Mojave and and Atacama Atacama deserts desertswith with aa liquid-phaseliquid-phase Clark Clark-type- •− O2 electrodetype O2 electrode [49]. It is [49] initiated. It is initiated (in step (in a) step by thea) by addition the addition of 50 of nmoles 50 nmoles O2•− O2(simulating (simulating regolith regolith O2•−, •− •− representedO2 , represented as x O2•− asmoles) x O2 moles) in the absencein the absence or presence or presence of 45 of units 45 units Cu/ Zn-superoxideCu/Zn-superoxide dismutase dismutase (SOD), (SOD), and the concentration of released O2 (by the SOD-catalyzed dismutation reaction of O2•−) is and the concentration of released O2 (by the SOD-catalyzed dismutation reaction of O2•−) is recorded recorded (as reading1 Adism = ½ xO2; see Treatment A in text), which is equal to the second dismutation (as reading Adism = ⁄2xO2; see Treatment A in text), which is equal to the second dismutation reaction 2 2 2 2 reaction product1 H O (= ½ xH O ). In a subsequent step b, the addition of catalase (CAT) causes the product H2O2 (= ⁄2xH2O2). In a subsequent step b, the addition of catalase (CAT) causes the additional additional release of O2 (via the CAT-catalyzed decomposition of H2O2, the second product of O2•− release of O2 (via the CAT-catalyzed decomposition of H2O2, the second product of O2•− dismutation), dismutation), which is also recorded (as reading Adism/CAT = ¼ xO2 plus the already released ½ xO2; see 1 1 which is also recorded (as reading Adism CAT =2− xO plus the already released ⁄2xO ; see Treatment B Treatment B in text). If there are also metal/ O2 4or free2 H2O2 present in regolith (represented2 as yH2O2 in text). If there are also metal O 2 or free H O present in regolith (represented as yH O moles), moles), these are simulated in Figure2 − 1 by the 2addition2 of 40 nmoles H2O2 (in step c). These peroxides2 2 thesewill are also simulated be decomposed in Figure by1 bythe theCAT addition (added in of step 40 nmoles b) to ½ y HO2,2 Oand2 (in in thisstep case c). These, the total peroxides released will O2 also 1 be decomposedwill be recorded by thein step CAT c as (added reading inAdism/CATstep b (the) to sum⁄2yO ½2,x andO2 + ¼ inx thisO2 + ½ case,yO2). the total released O2 will be 1 1 1 recorded in step c as reading Adism/CAT (the sum ⁄2xO2 + 4 xO2 + ⁄2yO2). 4. OxR Assay Simulation Verification on Mars-Analog Regolith

Life 2019, 9, 70 6 of 16

4. OxR Assay Simulation Veriﬁcation on Mars-Analog Regolith

The OxR assay was performed using a semi-sealed liquid-phase O2 electrode with known concentrations of O2•− and H2O2, and in the presence/absence of Mars-like regolith from the Mojave (CIMA volcanic field) and the Atacama deserts. The assay was further validated on commercial sources 2 2 of metal salts of O2•− (KO2) and O2 − (Na2O2, CaO2, MgO2) in the presence of CO − and ClO4− (both are present in Martian regolith). Gamma-radiation experiments were performed to evaluate the stability of the OxR assay enzymes CAT and SOD against cosmic radiation [49]. The electrode reaction chamber was filled with 1 mL potassium (K)-phosphate-DTPA buffer (0.25 M K-phosphate buffer, pH 7.2, containing 10 mM DTPA) to which the assay reagents (O2•−, H2O2, SOD, and CAT) were added at constant room temperature (RT). As already noted, DTPA chelates any soluble transition metal ions that can destroy H2O2 and O2•−. Moreover, DTPA will also prevent such chelated metals from inactivating the OxR assay protein reagents SOD and CAT via their oxidation by •OH (produced by way of the Fenton reaction) or via direct inhibition. The OxR assay was experimentally tested with known concentrations of O2•− and H2O2 added in the Clark-type O2 electrode, as illustrated in Figure1[ 49]. To validate the OxR assay enzymatic reactions 1 and 7 (in Table2) in the Clark-type O2 electrode chamber, the following treatments were performed (data are shown in Figure1): Treatment A (reaction 1, see Figure1 step a): SOD-catalyzed dismutation of O2•− to O2 and H2O2). Seventy microliters of O2− stock solution was added (final 50 µMO2•− or 50 nmoles) to the O2 electrode chamber, which contained 1 mL K-phosphate-DTPA buffer and 45 units (U) SOD (10 µl of 1 ± a 4500 U ml− stock made in ddH2O), and the released O2 concentration was recorded until a plateau was reached. Treatment B (reaction 7 in Table2, see Figure1 step b): CAT-catalyzed conversion to O2 of H2O2 released via SOD-catalyzed dismutation O2•− derived by O2•− hydrolysis, and H2O2 released from -1 peroxides via hydrolysis. After measuring the 1st O2(g) plateau (Treatment A), 20 U ml CAT (10 µl 1 2000 U ml− stock) was added to the resulting reaction mixture, and after the 2nd O2(g) plateau was reached, 10 µl 4 mM H2O2 (final 40 µM or 40 nmoles) was added, and the 3rd O2(g) plateau was recorded (Figure1 step c). Mathematical treatment of the data derived from treatments A and B: Assuming the presence of x O2•− and yH2O2 moles in the K-phosphate-DTPA buffer in the Clark O2 electrode chamber, these supero/peroxidants were calculated from the experiments illustrated in Figure1 as follows. The released O2 concentrations measured by the O2 electrode during treatments A and B (designated Adism and Adism/CAT, respectively) are described by the following molar equations and are based on the molar stoichiometry of the reactions 1 and 7 (in Table2): 1 Adism = ⁄2xO2; 1 simplified: Adism = ⁄2x, where x is O2•− moles

1 1 1 Adism/CAT = ⁄2xO2 + 4 xO2 + ⁄2yO2; 3 simpliﬁed: A = x + 1⁄2y, where y is H O moles dism/CAT 4 2 2

The molar concentrations of xO2•− and yH2O2 are then estimated using the following mathematical equations, derived by appropriately combining the molar equations Adism and Adism/CAT:

O2•− moles (= x) = 2Adism

H O moles (= y) = 2A 3A 2 2 dism/CAT − dism The released O2 concentrations (Adism and Adism/CAT) during the OxR assay (shown in Figure1) matched the concentrations predicted by the stoichiometry of each of the assay reactions 1 and 7. Indeed, when the values Adism (corresponding to 24 nmoles from step a) and Adism/CAT (corresponding to Life 2019, 9, 70 7 of 16

37 nmoles from step b, or 56 nmoles from steps b plus c) are inserted to the above molar equations for O2•− and H2O2, their calculated experimental concentrations are statistically equal to their concentrations which were added in the O2 electrode chamber. Simulation of cosmic radiation effect on the OxR assay enzymic reagents: Another consideration for the OxR instrument is whether its enzymatic reagents SOD and CAT would be functional upon exposure to cosmic radiation levels expected during missions to Mars, the Moon, and possibly icy satellites Jupiter and Saturn. To address this question for Mars and Moon, cosmic radiation simulation experiments were performed [49], where solid SOD and CAT were exposed to γ-radiation at a dose range comparable to that which would be received during a space mission. Activities were also determined for these enzymes in various concentrations (% v/v) of ACN since 100% ACN is used to wet the regolith sample to quantitatively purge out any unknown source trapped O2. The SOD retained functional activity after exposure to a γ-radiation dose of 6 Gy (an equivalent to the cosmic radiation dose received from 38 round trips to Mars [53]). The CAT specific activity was unaffected up to ~3 Gy (equivalent to 19 round trips to Mars, and many more trips to the Moon) after which it decreased linearly to 40% (of its unexposed activity) at 6 Gy. SOD activity was unaffected in up to 50% ACN, while CAT activity decreased in a manner that matched the ACN concentration. For example, an initial CAT specific 1 activity of ~3 U µg− at 0% ACN decreased by 50-fold at the maximum tested 50% ACN. This result 1 indicates that for an OxR assay based on 3 U µg− CAT at 0% ACN in laboratory testing, the CAT 1 concentration should be increased 50-fold (i.e., 150 U µg− ) plus a margin for flight, if a 50% ACN concentration is optimum for instrument implementation.

5. The Potential of the OxR Assay for a Field-Deployable Instrument The OxR assay can be extended to the search of possible metal supero/peroxidant cycles in terrestrial and extraterrestrial ecosystems. We expect that the full instrument can be packaged in 1 U (i.e., CubeSat sized at 10 cm/side) using a reaction chamber scheme with an O2-sensor (to monitor the enzymatic 2 release of O2 from O2•− and O2 − in a regolith sample during interaction (under constant mixing) with SOD and CAT, as illustrated and described in Figure2. During operation, the first step of mixing the regolith with anhydrous ACN is very crucial for the following reasons: ACN (actually containing 0.2 mM dicyclohexano-18-crown-6 ether, CE) flushes loosely bound O2 from unknown sources (designated zO2) necessary for instrument calibration at the same time the CE component, will facilitate O2•− dissociation from superoxo metal salts [1,54,55], and together they stabilize O2•− for the subsequent enzymic steps 2 and 3 (Figure2). In other words, the ACN-CE solvent used in step 1 prevents the dismutation of regolith O2•− to O2 that would occur with the use of an aqueous solvent (see reaction 1 in Table1), which would make the determination of background zO2 (and, thus, of x O2•− and yH2O2 moles) impossible. It should also be noted that although the OxR assay can quantify O2 released from unknown sources (i.e., zO2), it cannot discriminate the H2O2 generated by the hydrolysis of metal superoxide radicals and peroxides from that of any free H2O2 (possibly existing in mineral pore spaces). The accurate quantification of metal superoxide radicals and peroxides by the OxR requires that their initial hydrolysis products O2•− and H2O2, respectively, remain stable for SOD and CAT treatment. It has been already noted, the metal chelator DTPA and the phosphate buffer reagents will scavenge inorganic cations that affect the stability of O2•− and H2O2. Even if a fraction of H2O2 converts to O2 by factors other than CAT (e.g., by high 2 CO − concentration and high regolith alkalinity [49], or by the catalysts MnO2 [56], or silver, platinum, lead, ruthenate, and RuO2, which decompose H2O2 to O2 in alkaline solution [57]), this will not affect the accurate determination of metal superoxide radicals and peroxide concentrations since these factors will complement the conversion of H2O2 to O2 by CAT. Life 2019, 9, x FOR PEER REVIEW 8 of 16 also determined for these enzymes in various concentrations (% v/v) of ACN since 100% ACN is used to wet the regolith sample to quantitatively purge out any unknown source trapped O2. The SOD retained functional activity after exposure to a γ-radiation dose of 6 Gy (an equivalent to the cosmic radiation dose received from 38 round trips to Mars [53]). The CAT specific activity was unaffected up to ~3 Gy (equivalent to 19 round trips to Mars, and many more trips to the Moon) after which it decreased linearly to 40% (of its unexposed activity) at 6 Gy. SOD activity was unaffected in up to 50% ACN, while CAT activity decreased in a manner that matched the ACN concentration. For example, an initial CAT specific activity of ~3 U µg-1 at 0% ACN decreased by 50-fold at the maximum tested 50% ACN. This result indicates that for an OxR assay based on 3 U µg−1 CAT at 0% ACN in laboratory testing, the CAT concentration should be increased 50-fold (i.e., 150 U µg−1) plus a margin for flight, if a 50% ACN concentration is optimum for instrument implementation.

Figure 2. Diagrammatic principle of an OxR assay-based field instrument for the identification/ Figurequantification 2. Diagrammatic of regolith superoxide principle radicals of an and OxR peroxides assay-based (shown field as x O instrument2•− and yH 2 forO2 moles, the identification/quantificationrespectively): Regolith sample of regolith is subjected superoxide to the following radicals and released peroxides O2 recording (shown procedures.as xO2•− and In yH step2O2 1,

moles,the regolith respectively): is wetted Regolith with anhydroussample is subjected ACN to to flush the outfollowing loosely released bound O22 recording(designated procedures.zO2 moles; Inenclosed step 1, the in regolith dotted squares) is wetted for with (i) canceling anhydrous out ACN any to background flush out loosely O2 and bound (ii) measuring O2 (designated it as coming zO2 moles;from enclosed unidentified in dotted sources. squares) In step for 2,(i) SODcanceling is administered out any background in the instrument O2 and (ii) chamber measuring dissolved it as comingin K-phosphate-DTPA from unidentified buff er sources. (pH = 7.2)In step at an 2, equal SOD (at is least) administered to ACN volume in the (resulting instrument in at chamber least 50% dissolvedACN), and in K the-phosphate released O-DTPA2 (enclosed buffer in (pH solid-line = 7.2) at square, an equal which (at least) results to from ACN the volume group (resulting of metal O in2•− atvia least SOD-catalyzed 50% ACN), and dismutation the released of their O2 (enclosed hydrolysis in productsolid-line O 2square,•−, together which with results H2O from2) is recordedthe group by •− A •− ofthe metal chamber O2 via O2 SODsensor-catalyzed as reading dismutationdism. In a of subsequent their hydrolysis third step, product K-phosphate-DTPA-bu O2 , together withff Hered2O2) CAT is is introduced in the same chamber, where the additional released O (from the decomposition of H O recorded by the chamber O2 sensor as reading Adism. In a subsequent2 third step, K-phosphate-DTPA2 - 2 2 coming from the hydrolysis of both groups of metal O2•− and O2 −) is summed to that released from step 2 (and enclosed in three solid-line squares), and is recorded as reading Adism/CAT. The values of Adism and Adism/CAT (their net values determined by the experimental values designated by the arrows pointing at them on the Y-axis) are then used to determine the moles of regolith O2•− and H2O2, using their respective equations: O2•− = 2Adism (= x), and H2O2 = 2Adism/CAT - 3Adism (derived as shown in Section4, ‘OxR assay simulation verification on Mars-analog regolith’).

Non-enzymatic OxR instrument version: We have also developed a non-enzymatic OxR assay for cases where enzymatic stability may be insuﬃcient (e.g., missions to Titan, Europa, and Enceladus) or when the required long-term 20 C SOD and CAT storage is not possible. Moreover, some future − ◦ rovers may long outlast their expected life times (as past ones have done), and for whichever rover carries an OxR instrument the reagent enzymes may degrade over the years, whereas the inorganics may be more durable. A non-enzymatic version of the OxR instrument is based on the following reagents, which we have preliminarily tested successfully (data not shown): In place of SOD, CuSO4, MnCl2, and MnSO4 can be used: 1. CuSO4 (at 0.1 to 300 µM) and MnCl2 (at 0.1 to 100 µM)) [58,59]; MnCl2 dismutates O2•− as eﬀectively as SOD does [59]. Life 2019, 9, 70 9 of 16

2. MnSO (at 0.1 mM) has a rate constant for O dismutation k = 2.3 106 M 1 s 1 (in 5 mM 4 2•− × − − HEPES, pH = 7.8) [52]. This is 10-fold higher than the rate constant for the spontaneous aqueous dismutation of O (2 O + 2 H O 2 OH + H O + O ; k = 2x105 M 1 s 1 at pH = 7.8 [60]). 2•− 2•− 2 → − 2 2 2 − − In place of CAT, the following inorganic reagents can be used: 1. MnO2 acts as CAT-mimetic (2H2O2 2H2O + O2)[56]. 3 → 2. Ferricyanide [Fe(CN)6 −; FECN]. FECN reacts with H2O2 at a different stoichiometry than 3 4 + that of its CAT-catalyzed decomposition [1⁄2yH O + Fe(CN) Fe(CN) + H + 1⁄2yO [61,62]]. 2 2 6 − → 6 − 2 However, the use of FECN modifies the set of the equations for the determination of O2•− and H2O2 via released O2 (specifically the equation for H2O2). These are the following, designating as Adism/FECN as the reading value (by the O2-electrode) for the released O2 after treatment with FECN:

O2•− moles = 2Adism (same as with the enzymatic version of the OxR instrument)

H2O2 moles = A 2A dism/FECN − dism Concluding, the principle of the OxR assay can be used for the development of an instrument for 2 the detection of planetary and terrestrial O2•− and O2 − with the following considerations: 1. OxR assay enzymes SOD and CAT are used in excess; they are sufficient when used even in the amount of a few activity units. 2. SOD and CAT are stored (below 20 C for long-term storage) separate from their aqueous − ◦ solvents, and are mixed right before administration. This can be accomplished by storing them, for example, in two separate reagent crucibles (analogous to those used in the WCL instrument of the 2007 Phoenix Mars Scout Lander mission [63]), or in (commercially available) dual-chamber pre-fillable syringes (one chamber for storing the enzyme and one for its solvent, to be mixed upon piston movement), followed by their sequential dispensing in the instrument’s regolith chamber (under continuous mixing of its reagents). 3. The instrument can use solid state electrochemical or optical sensing O2-electrodes of high sensitivity. There are commercially available O2 probes (e.g., sensor type PSt6, by PreSens Precision Sensing GmbH, Regensburg, Germany) that are based on the luminescence quenching by O2, and are -3 sensitive enough to measure O2 at much lower concentrations (~1 nmole O2 cm regolith) than that (775 nmoles) detected by the GEX [26]. For example, the typical detection limit of the PreSens sensor PSt6 is 0.002% O2, with 1 ppb and 0.5 ppm for aqueous and gaseous O2, respectively. The PreSens Precision Sensing O2 probes come either as needle-type optical fiber probes (with a tip size < 50 µm, protected, e.g., inside a stainless-steel needle), or as implantable probes (with a tip size of < 50 to 140 µm, while the outer diameter ranges from 140 µm to 900 µm). Therefore, O2 sensing by the OxR instrument with solid-state sensors can be done in both gaseous and liquid phase. Regarding released O2 partition between liquid and headspace in the sample chamber, underestimation of the released reactive O2 due to such exsolution can be addressed by either adding an extra gas phase O2 sensor, or by the calculation of the partition between liquid and gas phase at the set pressure and temperature. 4. Respective ACN and SOD reagent process steps 1 and 2 are omitted in testing water samples (e.g., from Enceladus and Europa) by the OxR instrument, because O2•− dismutates to H2O2 and O2 under aqueous conditions (see reaction 1, Table1). In such an application, the first step of the OxR instrument will record O2 of unknown origin (for instrument calibration) in a melted ice sample. Following this step, CAT will be administered to convert to O2 any present H2O2. This will be the only peroxidant specifically determined by the OxR instrument in the (melted) ice samples from the surface or plums of Enceladus and Europa.

6. Implementation of the OxR Instrument One approach for implementing the OxR assay for field instrument construction is to keep it compatible with the Wet Chemistry Laboratory (WCL) that flew as part of the Phoenix lander mission to Mars [30]. The WCL (Figure3) consists of a lower beaker containing sensors designed to analyze the Life 2019, 9, 70 10 of 16 chemical properties of the regolith and an upper actuator assembly for adding regolith, water, reagents, and stirring [63]. The WCL sensor set included an O2 electrode, pressure sensor, and thermocouple needed for the OxR assay. A key part of the WCL system is the storage of liquid and dry reagents. Our prototype design uses a reagent dispenser assembly similar to WCL (which uses five crucibles to store the reagents to be dispensed). We will use the following three crucibles: A crucible for dispensing into the beaker the anhydrous ACN to wash out from regolith any background O2 (for recording its level). A crucible divided into two compartments to store the SOD enzyme and its solvent (for recording O2 released from the dismutation of regolith superoxide radicals; H2O2 will also be released by this dismutation) separately. A crucible divided into two compartments to store the CAT enzyme and its solvent (for recording

OLife2 released2019, 9, x FOR from PEER superoxide REVIEW radical-derived H2O2, and that derived from regolith peroxides) separately.11 of 16

Figure 3. Image of the Wet ChemistryChemistry LaboratoryLaboratory (WCL)(WCL) fromfrom thethe PhoenixPhoenix Lander.Lander.

TheThe automated automated and and sequential sequential dispensing dispensing of the of reagents the reagents is critical is criticalto the success to the of success the prototype. of the Aprototype. diagram ofA thediagram Phoenix of systemthe Phoenix is shown system in Figure is shown4. The in reagent Figure dispenser4. The reagent assembly dispenser will be assembly coupled towill the be construction coupled to andthe construction operational testingand operational of the beaker testing (reaction of the cell). beaker A diagram(reaction of cell). the WCLA diagram reaction of cellthe WCL is shown reaction in Figure cell is5 .shown In contrast in Figure to the 5. In complex contrast array to the of complex sensors array in the of WCL sensors on in Phoenix the WCL [ 63 on], wePhoenix have only[63], Ow2esensors, have only temperature, O2 sensors, and temperature pressure. Joining, and pressure. the reagent Joining dispenser the reagent assembly dispenser and the reactionassembly cell and completes the reaction the prototype.cell completes However, the prototype. the WCL However, instrument the uses WCL a 25-cc instrument chamber uses to analyze a 25-cc 1chamber cc of regolith. to analyze For some 1 cc missions, of regolith. this For is an some important missions issue, this and is motivates an important microfluidics issue and approaches. motivates Anmicrofluidics alternative approaches.instrument construction An alternative approach instrument will be construction based on the approach microfluidic will transport be based/delivery on the technologymicrofluidic [64 transport/delivery–69], already developed technology by the [64 R.A.–69], Mathies’s already developed Space Sciences by the Laboratory R.A. Mathies’s at Berkeley Space University.Sciences Laboratory The chip for at microscopic Berkeley University. fluid transport The chip between for microscopic the components fluid of transport an instrument between such the as OxRcomponents (e.g., reagent of an storage instrument capsules, such regolith as OxR sample (e.g., reagent chamber storage with O capsules,2/temp/pressure regolith sensors, sample and chamber waste reservoirs),with O2/temp/pressure is analogous sensors, to digital and electronic waste reservoirs), processors, andis analogous all that is to needed digital is elect a changeronic inprocessors, the order ofand operations all that is conducted needed is by a the change device in [66 the]. order All macroscopic of operations reagent conducted volumes by are the contained device [66] within. All stainlessmacroscopic steel reagent bellows volumes expanded are orcontained contracted within by externallystainless steel applied bellows N2 expandedgas. The OxRor contracted instrument by chipexternally can be applied constructed N2 gas as. a The scaled-down OxR instrume versionnt chip (e.g., can a 200 be gr, constructed 2 Watts, 10x10x10 as a scaled cm package)-down version of the Enceladus(e.g., a 200 Organicgr, 2 Watts, Analyzer 10x10x10 (EOA) cm chippackage) (Figure of 6the). Enceladus Organic Analyzer (EOA) chip (Figure 6). The OxR instrument prototype will be tested in a laboratory setting and results compared to standardThe OxR laboratory instrument procedures, prototype followedwill be tested by field in a testing laboratory in the setting Mojave and Desert. results This compared has been to astandard continuing laboratory test site for procedures, our studies followed [1], and, by thus, field we testing have a deepin the knowledge Mojave Desert. base of This the site has and been the a expectedcontinuing results test site providing for our astudies convenient [1], and, basis thus, for prototypewe have a testing.deep knowledge base of the site and the expected results providing a convenient basis for prototype testing.

Figure 4. Diagram of the reagent dispenser assembly with crucibles ready for deployment [63].

Life 2019, 9, x FOR PEER REVIEW 11 of 16

Figure 3. Image of the Wet Chemistry Laboratory (WCL) from the Phoenix Lander.

The automated and sequential dispensing of the reagents is critical to the success of the prototype. A diagram of the Phoenix system is shown in Figure 4. The reagent dispenser assembly will be coupled to the construction and operational testing of the beaker (reaction cell). A diagram of the WCL reaction cell is shown in Figure 5. In contrast to the complex array of sensors in the WCL on Phoenix [63], we have only O2 sensors, temperature, and pressure. Joining the reagent dispenser assembly and the reaction cell completes the prototype. However, the WCL instrument uses a 25-cc chamber to analyze 1 cc of regolith. For some missions, this is an important issue and motivates microfluidics approaches. An alternative instrument construction approach will be based on the microfluidic transport/delivery technology [64–69], already developed by the R.A. Mathies’s Space Sciences Laboratory at Berkeley University. The chip for microscopic fluid transport between the components of an instrument such as OxR (e.g., reagent storage capsules, regolith sample chamber with O2/temp/pressure sensors, and waste reservoirs), is analogous to digital electronic processors, and all that is needed is a change in the order of operations conducted by the device [66]. All macroscopic reagent volumes are contained within stainless steel bellows expanded or contracted by externally applied N2 gas. The OxR instrument chip can be constructed as a scaled-down version (e.g., a 200 gr, 2 Watts, 10x10x10 cm package) of the Enceladus Organic Analyzer (EOA) chip (Figure 6). The OxR instrument prototype will be tested in a laboratory setting and results compared to standard laboratory procedures, followed by field testing in the Mojave Desert. This has been a continuingLife 2019, 9, 70 test site for our studies [1], and, thus, we have a deep knowledge base of the site and11 ofthe 16 expected results providing a convenient basis for prototype testing.

Life 2019, 9, x FOR PEER REVIEW 12 of 16

FigureFigureLife 4. 4. 2019 DiagramDiagram, 9, x FOR PEERof of the the REVIEW reagent reagent dispenser dispenser assembly assembly with with crucibles crucibles ready ready for deployment 12 of 16 [63]. [ 63].

Figure 5. Diagram of the WCL reaction cell showing water storage and stirring rod. Figure 5. DiagramFigure 5. Diagram of the of WCL the WCL reaction reaction cell cell showingshowing water water storage storage and stirring and stirringrod. rod.

Figure 6. Diagram of the Enceladus Organic Analyzer (EOA) data programmable chip (modified from [65]).

7. Studies with the OxR Instrument Figure 6. Diagram of the Enceladus Organic Analyzer (EOA) data programmable chip (modified from [65]). Figure 6. DiagramThe OxR of instrumentthe Enceladus can have Organic the following Analyzer potential (EOA) applications: data programmable chip (modified from [65]). 2•− 22− 7. Studies withIdentification the OxR Instrument of the ROS O and O , on the Moon and Mars, with extension to future missions to Jupiter’s satellite Europa and Saturn’s Enceladus and Titan. 7. StudiesThe OxR with instrument theMonitor OxR the Instrument canlevels have of ROS the for following astronaut health potential and safety, applications: given that O2•− can become biotoxic 3+ 2+ 2+ + •− (via conversion of Fe /Cu to Fe /Cu2, which, via the Fenton-reaction with the other ROS O2 , will Identiﬁcation of the ROS O and O ,• on the Moon and Mars, with extension to future missions The OxRgenerate instrument the highly can biotoxic have2•− freethe radicalfollowing2 − OH [44]).potential Moreover, applications measuring dust/silica-induced: ROS to Jupiter’s satellitegeneration Europa is crucial andfor the Saturn’s evaluation Enceladus of possible health and Titan.hazards [18] in future manned missions to Identification of the ROS O2•− and O22−, on the Moon and Mars, with extension to future missions Mars and Moon. Monitor the levels of ROS for astronaut health and safety, given that O2•− can become biotoxic to Jupiter’s satelliteIdentify Europa3+ mineral2 and+ deposits Saturn’s2 +rich in +Enceladus ROS to be used and as Titanan O2g .source for human consumption. O2g (via conversion of Fe /Cu to Fe /Cu , which, via the Fenton-reaction•− with the other ROS O2•−, Monitorcan the be levelseasily produced of ROS on for a large astronaut scale due health to the following and safety ROS ,reactions: given that O2 isO converted2•− can become to O2g biotoxic will generateby the mixing highly with biotoxic (i) H2O (also free releasing radical H•2OOH2), or [ 44(ii)]). Fe3+ Moreover, or Cu2+ [44]. measuringO2g can be produced dust/silica-induced from O22− ROS (via conversion of Fe3+/Cu2+ to Fe2+/Cu+, which, via the Fenton-reaction with the other ROS O2•−, will generation is(e.g., crucial H2O2 foralso the released evaluation from reaction of possible (i)) by mixing health with hazards MnO2 [56] [18 ] or in silver, future platinum, manned lead, missions to generate the highly biotoxic free radical •OH [44]). Moreover, measuring dust/silica-induced ROS Mars and Moon.ruthenate, or RuO2 [57]. generation is crucialIdentify for O 2the•−/O 2evaluation2− on the metal partsof possible of manned health space vehicles/stations. hazards [18] ROSin future can be generatedmanned by missions to Identify mineral deposits rich in ROS to be used as an O •source− 2− for human consumption. O Mars and Moon.a combination of O2g (in vehicle) with cosmic radiation [1]. 2g O2 and O2 have implications for 2g can be easilyexploration produced because on a they large can: scale due to the following ROS reactions: O2•− is converted to O2g 2g 2g Identify mineral(i) cause deposits corrosive oxidativerich in ROSdeterioration to be ofused space as vehicles/stations, 3a+n O source2+ for human consumption. O by mixing with (i) H2O (also releasing H2O2), or (ii) Fe or Cu [44]. O2g can be produced from can be easily produced(ii) pose aon serious a large risk scale for oxidative due to modification the following of stored ROS foods, reactions: making themO2•− unsafeis converted for to O2g by mixing withastronauts, (i) H2 O (also releasing H2O2), or (ii) Fe3+ or Cu2+ [44]. O2g can be produced from O22− (iii) compromise astronaut health due to their well-known biotoxic effects [44]. (e.g., H2O2 also released from reaction (i)) by mixing with MnO2 [56] or silver, platinum, lead, ruthenate, or RuO2 [57]. Identify O2•−/O22− on the metal parts of manned space vehicles/stations. ROS can be generated by a combination of O2g (in vehicle) with cosmic radiation [1]. O2•− and O22− have implications for exploration because they can: (i) cause corrosive oxidative deterioration of space vehicles/stations, (ii) pose a serious risk for oxidative modification of stored foods, making them unsafe for astronauts, (iii) compromise astronaut health due to their well-known biotoxic effects [44].

Life 2019, 9, 70 12 of 16

2 O2 − (e.g., H2O2 also released from reaction (i)) by mixing with MnO2 [56] or silver, platinum, lead, ruthenate, or RuO2 [57]. 2 Identify O2•−/O2 − on the metal parts of manned space vehicles/stations. ROS can be generated 2 by a combination of O2g (in vehicle) with cosmic radiation [1]. O2•− and O2 − have implications for exploration because they can: (i) cause corrosive oxidative deterioration of space vehicles/stations, (ii) pose a serious risk for oxidative modification of stored foods, making them unsafe for astronauts, (iii) compromise astronaut health due to their well-known biotoxic effects [44]. Identify locations on Mars and the Moon with low ROS levels which may be indicative of the high potential for biosignature (e.g., [70,71]) preservation. Of particular upcoming interest is the Dragonfly mission to Titan by NASA (launched in 2026, and landing in 2034), which will search for evidence of prebiotic chemical processes on the surface of Titan [72]. The instrument is also applicable to terrestrial research, with indicative studies being: (i) O2•−/O2− association to microorganisms’ oxidative stress in extreme desert environments, with extension to life’s origin [1,73]; (ii) health hazard implications from measuring ROS-reactivity of (a) volcanic ash (due to •OH generation) [74], and (b) pyrites (from O2/H2O2/surface-bound ferric iron-induced •OH generation during pyrite oxidation) in coal mining regions [75].

8. Conclusions We have developed a sensitive assay for the use in a future Oxygen Release (OxR) instrument for the detection of ROS, with potential applications to the Mars, Moon, Europa, Titan, and Enceladus missions. The instrument can support the exploration of the Moon (including monitoring of astronaut health hazards), exploration on Mars, Moon, and Titan, and terrestrial studies. The OxR instrument is based on the selective and specific enzymatic decomposition of supero/peroxidants to O2 and their quantification by the measurement of the released O2. An alternative non-enzymatic option is also proposed. Laboratory simulations and the sensitivity of the commercially available O2 sensors indicate 2 2 that the OxR instrument can detect metal O2•−/O2 − in the Martian and Lunar regolith and also O2 − in the icy waters of the satellites of Saturn Enceladus and Titan (in its regolith too) and of Jupiter’s Europa, at levels as low as 10 ppb. In terms of Technology Readiness Level, the OxR instrument is at 3 (method validated in the lab), and can be made flight-ready by leveraging the Phoenix Wet Chemical Laboratory hardware or a microfluidic transport/delivery technology.

Author Contributions: OxR assay was C.D.G. experimentally developed with contribution by C.P.M., E.K., P.P., M.S.; OxR instrument conceptualization by C.D.G., R.C.Q., C.P.M.; Instrument’s prototype general design conceptualization by R.C.Q., C.P.M.,with contribution by C.D.G.; Writing of original draft by C.D.C.; Writing/review & editing by C.D.C., C.P.M., R.C.Q. Funding: This research received no external funding. Acknowledgments: C.D.G. acknowledges the support of the Greek Ministry of Education, and R.C.Q. acknowledges the support of the NASA Astrobiology Institute (SETI Institute Team). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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